The project will be devoted to an analysis of the way isoleucine and valine metabolism is regulated in Escherichia coli. The involvement of the branched chain amino acyl tRNA synthetases (or the cognate tRNA's) in the control of termination at multiple sites in the ilvEDA operon will be examined. In vitro analysis of the way transcription initiation of the ilvEDA operon is controlled will be examined by following beta-galactosidase synthesis from an ilvED'-lacZ DNA template carried on a lambda phage genome. In vitro analysis of ilvC gene regulation will also be studied with an ilv-lac fusion that permits the induction of beta-galactosidase with the ilvC inducer, acetohydroxybutyrate. A factor required for this induction is being characterized. Our restriction enzyme analysis and heteroduplex mapping should enable us to distinguish between promoter-proximal and promoter-distal message fragments. The role of the ilvU locus in maturation of tRNAVal and tRNAIle will be determined.